In multicellular organisms certain peptides (“precursors”), like neuropeptides, are post-translationally modified in a series of enzymatic steps that cleave and further modify peptide substrates to yield fully functional bioreactive peptides. The process begins in the trans-Golgi apparatus and continues as immature secretory granules. A very important late stage post-translational modification for many of these peptides is carboxyterminal alpha amidation.
Alpha-amidation of C-terminal residues is pivotal for the activity of several peptide hormones involved in the human or animal metabolism. Several peptide hormones are today used as drugs in the treatment of humans, e.g. for control of obesity and/or diabetes, or are under development as potential drugs. An example of such a peptide hormone is amylin (e.g. Symlin®, pramlintide acetate, which is an analogue of human amylin). Human amylin is a 37 amino acid residue peptide which can be used for treating or preventing obesity and/or diabetes. Accordingly, the C-terminal of amylin needs to be amidated in order to obtain full biological activity. Likewise Peptide YY (PYY) should be alpha amidated to obtain full biological activity.
E. coli, and yeast are widely used for recombinant expression of peptides of eukaryote origin. However, due to the nature of the C-terminal α-amide group peptide hormones cannot be expressed in an active form using state of art microbial expression systems based on E. coli and yeast, as alpha amidating enzymes are not expressed naturally in these organisms. Therefore a C-terminal alpha amide must be introduced in the recombinantly expressed peptides using for an example an ex vivo modification with alpha amidating enzymes.
Enzymatic modification of peptide precursors with a C-terminal Gly to α-amide by means of a bifunctional peptidyl α-amidating monooxygenase (PAM) is found in several eukaryotic organisms. Multiple alternatively spliced transcript variants encoding different isoforms have been described for this enzyme. The enzyme has exclusively been described for multicellular organisms (Metazoa). The conversion of C-terminal Gly residue in a peptide to α-amide is a two-step process, where the N-terminal domain of PAM (named PHM) catalyses the conversion of Glycine to α-hydroxyglycine and the C-terminal domain of PAM (named PAL) catalysis the conversion of the α-hydroxyglycine to α-amide. In eukaryotic organisms the two catalytic domains work sequentially to catalyze neuroendocrine peptides to active alpha-amidated products. Two disulphidebridges are highly conserved in PAL domains from eukaryotic organisms.
While it may be possible to synthesize by chemical means small peptides which contain an amide group at the C-terminal end (alpha amide), larger alpha-amidated peptides are difficult and expensive to produce. Alpha amidating enzymes are thus useful in the conversion of recombinant precursor peptides to mature peptides.
U.S. Pat. No. 4,708,934 describes a peptidyl-glycine α-amidating monooxygenase extracted from medullary thyroid carcinoma cell lines and tissue samples.
U.S. Pat. No. 5,789,234 describes the production of an alpha-amidating enzyme by recombinant DNA techniques.
WO90/08194 relates to a process for production of C-terminal alpha amidated peptide from a precursor peptide by the use of a eukaryotic C terminal alpha amidating enzyme. Also described is a method for eukaryotic expression of these C terminal alpha amidating enzymes.
WO89/02460 describes a bovine derived PAM enzyme, its cloning, cDNA and expression by recombinant DNA technology.
EP0448513 describes a process for recombinant expression of a peptidylglycine alpha-hydroxylating monooxygenase derived from Xenopus Laevis, comprising culturing insect cells transfected with a recombinant baculovirus to which a DNA coding for the peptidylglycine alpha-hydroxylating monooxygenase has been incorporated to produce the enzyme.
EP0465404 describes an enzyme (PHL; PAL) derived from Xenopus Laevis catalysing the cleavage of the N—C bond in the α-hydroxylglycine moiety of a C-terminally α-hydroxylated peptide, the cloning of the enzyme and its recombinant expression in insect cells.
US 20060292672 describes a cell line for expressing PAM or one of its two catalytic domains.
EP2172550 describes a recombinant C-terminal alpha-amidating enzyme derivative which lack the formation of at one of the five disulfide bonds normally occurring in a C-terminal alpha-amidating enzyme derived from Xenopus laevis and method of producing said derivative recombinantly in E. coli where the inclusions body obtained is solubilized and subjected to a refolding procedure.